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Sister chromatid exchange in human lymphocytes exposed to isoflurane and nitrous oxide in vitro
British Journal of Anaesthesia 82 (2): 268–70 (1999)
Sister chromatid exchange in human lymphocytes exposed to isoflurane and nitrous oxide in vitro K. H. Hoerauf1*, K. F. Schro¨gendorfer1, G. Wiesner3, M. Gruber3, A. Spacek1, H.-G. Kress1 and H. W. Ru¨diger2 1Department
of Anaesthesiology and General Intensive Care (B) and 2Department of Occupational Medicine, University Hospital of Vienna, Wa¨hringer Gu¨rtel 18–20, A-1090 Vienna, Austria. 3Department of Anaesthesiology, University Hospital of Regensburg, Germany *To whom correspondence should be addressed The question of whether or not inhalation anaesthetics are genotoxic remains controversial. Therefore, we have studied the in vitro genotoxic potential of isoflurane and nitrous oxide in human lymphocytes. Blood samples were obtained from eight healthy male, non-smoking volunteers, which were incubated and exposed to increasing concentrations of isoflurane (0.0, 0.3, 0.6 and 1.2 mmol litre–1) or 50% nitrous oxide in oxygen. Baseline sister chromatid exchange (SCE) rate per cell was mean 7.65 (SD 1.5) which increased to 9.15 (1.0), 9.55 (1.4) and 9.95 (1.8) SCE/cell during exposure to isoflurane 0.3, 0.6 and 1.2 mmol litre–1, respectively. During 50% nitrous oxide exposure, SCE rate was 9.26 (1.4). The difference between the control and exposed cells was statistically significant (Pø0.05). We conclude that exposure to nitrous oxide and subanaesthetic concentrations of isoflurane can produce genetic damage in peripheral lymphocytes in vitro. Br J Anaesth 1999; 82: 268–70 Keywords: anaesthetics volatile, isoflurane; anaesthetics gases, nitrous oxide; genetic factors, anaesthetics Accepted for publication: September 2, 1998
Evidence of genotoxic effects of isoflurane and nitrous oxide is controversial. Some studies have been unable to demonstrate genotoxicity, while others observed DNA damage after in vivo exposure to isoflurane and nitrous oxide.1 Aside from patient exposure, operating room staff are also exposed, although to a much lower concentration but over a longer period of time. This matter is currently being discussed in relation to an increased rate of spontaneous abortion in operating room personnel.2 Currently, the available data are inconsistent and clear results from in vitro testing of inhalation anaesthetics in a human cell system are absent. The sister chromatid exchange (SCE) test is considered to be a sensitive end-point for detecting genotoxic potential of mutagenic and carcinogenic agents. Therefore, we have investigated the genotoxic effects of isoflurane and nitrous oxide on the frequency of SCE in human lymphocyte cell cultures.
Methods and results After obtaining approval from the Local Ethics Committee, we obtained heparinized venous blood samples from eight volunteers. To minimize the influence of possible confounding factors, all volunteers were healthy, non-smoking
males, aged 30–38 yr. Whole blood (0.5 ml) cultures were established in Teflon tubes (Merck, Austria) containing 5 ml of chromosome medium 1A (Gibco, Austria), 5-bromo-29deoxyuridine (Sigma, Germany) 50 µmol litre–1, phytohaemagglutinin 0.05 ml and increasing concentrations of isoflurane (Abbott, Austria). Aliquots of medium containing isoflurane 14.49 µmol ml–1 were added to aliquots of isoflurane-free medium to obtain the expected concentrations of 0.0, 0.3, 0.6 and 1.2 mmol litre–1. Mean loss of isoflurane measured using head-space gas chromatography (Hewlett-Packard, Germany) after 72 h was 8.960.6% at all concentrations. Additionally, one culture of each volunteer was exposed to nitrous oxide. In this case, air over the culture was mixed with 50% nitrous oxide and shaken for 1 h before starting the culture. Mean loss was 12.660.8%, which was measured using infrared spectrometry (Capnomac, Datex, Helsinki, Finland). The concentrations used corresponded to subanaesthetic and anaesthetic concentrations of isoflurane, and only one anaesthetic concentration of nitrous oxide. After 72 h, cells were harvested and washed, and the remaining lymphocytes were prepared and stained.3 All scoring and counting procedures were performed by the same investig-
© British Journal of Anaesthesia
Genotoxicity of inhaled anaesthetics
Comment
Fig 1 Sister chromatid exchanges per cell (SCE/cell) after exposure to increasing concentrations of isoflurane (Iso.) and a single concentration of nitrous oxide. Control (C) value was significantly different from all other values (*Pø0.05).
ator using a non-automated microscope. Cell cycle kinetics were examined by scoring the proportion of cells at M1 (metaphase cells in the first replication cycle), M2 and M3. The proliferative rate index (PRI) was calculated according to the formula: PRI5(13M1%123M2%133M3%) /100. The mitotic index (MI) was calculated as the proportion of metaphases among the total cell population by counting a total of 1000 cells. SCE were measured by examining 15 complete second metaphases, one SCE being counted each time two adjacent segments of one of the chromatids in a chromosome were stained differently. Based on data from previous reports,3 the intention was to detect a difference between groups of 1.5 SCE per cell (SD 1.3 SCE per cell), when scoring 15 metaphases per probe. An α-error of 5%, and a minium power of 70%, resulted in a study population of eight probands (nQuery for Windows 95). After confirming normal distribution ANOVA with the Student–Newman–Keuls test, correction for multiple comparisons was applied to determine significant differences between the treated cells with Pø0.05. Isoflurane and nitrous oxide produced an increase in the rate of SCE. The means differed significantly between control cells and those exposed to increasing concentrations of isoflurane or nitrous oxide (Fig. 1). In the presence of anaesthetics, PRI decreased from mean 1.84 (SD 0.2) (control) to 1.67 (0.3) (isoflurane 0.3 mmol litre–1), 1.60 (0.5) (isoflurane 0.6 mmol litre–1), 1.38 (0.3) (isoflurane 1.2 mmol litre–1) and 1.63 (0.1) (50% nitrous oxide). MI also decreased from 7.39 (4.9)% (control), to 6.75 (3.6)% (isoflurane 0.3 mmol litre–1), 5.01 (3.0)% (isoflurane 0.6 mmol litre–1), 2.37 (1.6)% (isoflurane 1.2 mmol litre–1) and 2.05 (0.8)% (50% nitrous oxide). However, only the difference between controls and the highest concentration of isoflurane was statistically significant. For nitrous oxide, PRI and MI decreased compared with controls, but only MI was significant.
Various screening tests use genetic damage as an indicator of potential mutagenicity. One of these, the Ames Salmonella bacterial assay, is based on reversion of a histidine-requiring strain of Salmonella typhimurium to a non-histidine-requiring mutant after exposure to mutagenic chemicals. In this test system, using a variety of bacterial strains and test conditions, nitrous oxide and isoflurane were not mutagenic.4 In contrast, the SCE test involves exchange of DNA segments between two sister chromatids in a chromosome during cell replication. An increased frequency of SCE indicates exposure to mutagenic substances. Using this test system, we demonstrated an increase in the SCE rate for isoflurane and nitrous oxide compared with controls. This controversial finding could be explained by several factors. First, the Ames and SCE tests have different endpoints for detecting mutagenicity. Although both examine alterations in DNA, in some cases the Ames test was unable to detect mutagens, which later proved to be mutagenic or to interact with DNA of mammalian cells.4 Second, the concentration and exposure time were different to previous studies. Husum detected an increase in SCE formation in smoking but not in non-smoking patients exposed to anaesthetic concentrations of isoflurane and nitrous oxide.5 Unfortunately, data concerning exposure time and concentrations of anaesthetics were not available and therefore dose–responses could not be established. Although it is likely that exposure time was longer in our study, we demonstrated an increase in SCE in peripheral blood lymphocytes exposed to inhalation anaesthetics. Currently, we cannot conclude that there is any indication for the development of increased SCE rates in patients receiving clinical concentrations of isoflurane and nitrous oxide for short surgical procedures. Although the observed SCE rates were small compared with alkylating mutagens and carcinogens, it should be noted that an increase of approximately 1 SCE per cell is comparable with smoking 11–20 cigarettes a day.5 From our results, it seems unlikely that chronic exposure results in an increased SCE rate as the concentrations used in our study could not be achieved under normal operating room working conditions.6 Longterm investigations in operating room personnel exposed to different levels of waste anaesthetic gases are needed to clarify any potential genotoxicity of anaesthetic gases.
References
269
1 Reitz M, Antonini-Rumpf E, Lanz F. DNA single strand breaks in peripheral human lymphocytes after anaesthesia with isoflurane– nitrous oxide oxygen. Arzneimittelforschung 1993; 43: 1258–61 2 Boivin J. Risk of spontaneous abortion in women occupationally exposed to anaesthetic gases: a meta-analysis. Occup Environ Med 1997; 54: 541–8
Hoerauf et al.
3 Holz O, Scherer G, Brodtmeier S, et al. Determination of low level exposure to volatile aromatic hydrocarbons and genotoxic effects in workers at a styrene plant. Occup Environ Med 1995; 52: 420–8 4 Waskell L. A study of the mutagenicity of anesthetics and their metabolites. Mutat Res 1978; 57: 141–53
270
5 Husum B. Mutagenicity of inhalation anaesthetics studied by the sister chromatid exchange test in lymphocytes of patients and operating room personnel. Dan Med Bull 1987; 34: 159–70 6 Hoerauf K, Koller C, Jakob W, Taeger K, Hobbhahn J. Isoflurane waste gas exposure during general anaesthesia: the laryngeal mask compared with tracheal intubation. Br J Anaesth 1996; 77: 189–93
Sister chromatid exchange in human lymphocytes exposed to isoflurane and nitrous oxide in vitro K. H. Hoerauf1*, K. F. Schro¨gendorfer1, G. Wiesner3, M. Gruber3, A. Spacek1, H.-G. Kress1 and H. W. Ru¨diger2 1Department
of Anaesthesiology and General Intensive Care (B) and 2Department of Occupational Medicine, University Hospital of Vienna, Wa¨hringer Gu¨rtel 18–20, A-1090 Vienna, Austria. 3Department of Anaesthesiology, University Hospital of Regensburg, Germany *To whom correspondence should be addressed The question of whether or not inhalation anaesthetics are genotoxic remains controversial. Therefore, we have studied the in vitro genotoxic potential of isoflurane and nitrous oxide in human lymphocytes. Blood samples were obtained from eight healthy male, non-smoking volunteers, which were incubated and exposed to increasing concentrations of isoflurane (0.0, 0.3, 0.6 and 1.2 mmol litre–1) or 50% nitrous oxide in oxygen. Baseline sister chromatid exchange (SCE) rate per cell was mean 7.65 (SD 1.5) which increased to 9.15 (1.0), 9.55 (1.4) and 9.95 (1.8) SCE/cell during exposure to isoflurane 0.3, 0.6 and 1.2 mmol litre–1, respectively. During 50% nitrous oxide exposure, SCE rate was 9.26 (1.4). The difference between the control and exposed cells was statistically significant (Pø0.05). We conclude that exposure to nitrous oxide and subanaesthetic concentrations of isoflurane can produce genetic damage in peripheral lymphocytes in vitro. Br J Anaesth 1999; 82: 268–70 Keywords: anaesthetics volatile, isoflurane; anaesthetics gases, nitrous oxide; genetic factors, anaesthetics Accepted for publication: September 2, 1998
Evidence of genotoxic effects of isoflurane and nitrous oxide is controversial. Some studies have been unable to demonstrate genotoxicity, while others observed DNA damage after in vivo exposure to isoflurane and nitrous oxide.1 Aside from patient exposure, operating room staff are also exposed, although to a much lower concentration but over a longer period of time. This matter is currently being discussed in relation to an increased rate of spontaneous abortion in operating room personnel.2 Currently, the available data are inconsistent and clear results from in vitro testing of inhalation anaesthetics in a human cell system are absent. The sister chromatid exchange (SCE) test is considered to be a sensitive end-point for detecting genotoxic potential of mutagenic and carcinogenic agents. Therefore, we have investigated the genotoxic effects of isoflurane and nitrous oxide on the frequency of SCE in human lymphocyte cell cultures.
Methods and results After obtaining approval from the Local Ethics Committee, we obtained heparinized venous blood samples from eight volunteers. To minimize the influence of possible confounding factors, all volunteers were healthy, non-smoking
males, aged 30–38 yr. Whole blood (0.5 ml) cultures were established in Teflon tubes (Merck, Austria) containing 5 ml of chromosome medium 1A (Gibco, Austria), 5-bromo-29deoxyuridine (Sigma, Germany) 50 µmol litre–1, phytohaemagglutinin 0.05 ml and increasing concentrations of isoflurane (Abbott, Austria). Aliquots of medium containing isoflurane 14.49 µmol ml–1 were added to aliquots of isoflurane-free medium to obtain the expected concentrations of 0.0, 0.3, 0.6 and 1.2 mmol litre–1. Mean loss of isoflurane measured using head-space gas chromatography (Hewlett-Packard, Germany) after 72 h was 8.960.6% at all concentrations. Additionally, one culture of each volunteer was exposed to nitrous oxide. In this case, air over the culture was mixed with 50% nitrous oxide and shaken for 1 h before starting the culture. Mean loss was 12.660.8%, which was measured using infrared spectrometry (Capnomac, Datex, Helsinki, Finland). The concentrations used corresponded to subanaesthetic and anaesthetic concentrations of isoflurane, and only one anaesthetic concentration of nitrous oxide. After 72 h, cells were harvested and washed, and the remaining lymphocytes were prepared and stained.3 All scoring and counting procedures were performed by the same investig-
© British Journal of Anaesthesia
Genotoxicity of inhaled anaesthetics
Comment
Fig 1 Sister chromatid exchanges per cell (SCE/cell) after exposure to increasing concentrations of isoflurane (Iso.) and a single concentration of nitrous oxide. Control (C) value was significantly different from all other values (*Pø0.05).
ator using a non-automated microscope. Cell cycle kinetics were examined by scoring the proportion of cells at M1 (metaphase cells in the first replication cycle), M2 and M3. The proliferative rate index (PRI) was calculated according to the formula: PRI5(13M1%123M2%133M3%) /100. The mitotic index (MI) was calculated as the proportion of metaphases among the total cell population by counting a total of 1000 cells. SCE were measured by examining 15 complete second metaphases, one SCE being counted each time two adjacent segments of one of the chromatids in a chromosome were stained differently. Based on data from previous reports,3 the intention was to detect a difference between groups of 1.5 SCE per cell (SD 1.3 SCE per cell), when scoring 15 metaphases per probe. An α-error of 5%, and a minium power of 70%, resulted in a study population of eight probands (nQuery for Windows 95). After confirming normal distribution ANOVA with the Student–Newman–Keuls test, correction for multiple comparisons was applied to determine significant differences between the treated cells with Pø0.05. Isoflurane and nitrous oxide produced an increase in the rate of SCE. The means differed significantly between control cells and those exposed to increasing concentrations of isoflurane or nitrous oxide (Fig. 1). In the presence of anaesthetics, PRI decreased from mean 1.84 (SD 0.2) (control) to 1.67 (0.3) (isoflurane 0.3 mmol litre–1), 1.60 (0.5) (isoflurane 0.6 mmol litre–1), 1.38 (0.3) (isoflurane 1.2 mmol litre–1) and 1.63 (0.1) (50% nitrous oxide). MI also decreased from 7.39 (4.9)% (control), to 6.75 (3.6)% (isoflurane 0.3 mmol litre–1), 5.01 (3.0)% (isoflurane 0.6 mmol litre–1), 2.37 (1.6)% (isoflurane 1.2 mmol litre–1) and 2.05 (0.8)% (50% nitrous oxide). However, only the difference between controls and the highest concentration of isoflurane was statistically significant. For nitrous oxide, PRI and MI decreased compared with controls, but only MI was significant.
Various screening tests use genetic damage as an indicator of potential mutagenicity. One of these, the Ames Salmonella bacterial assay, is based on reversion of a histidine-requiring strain of Salmonella typhimurium to a non-histidine-requiring mutant after exposure to mutagenic chemicals. In this test system, using a variety of bacterial strains and test conditions, nitrous oxide and isoflurane were not mutagenic.4 In contrast, the SCE test involves exchange of DNA segments between two sister chromatids in a chromosome during cell replication. An increased frequency of SCE indicates exposure to mutagenic substances. Using this test system, we demonstrated an increase in the SCE rate for isoflurane and nitrous oxide compared with controls. This controversial finding could be explained by several factors. First, the Ames and SCE tests have different endpoints for detecting mutagenicity. Although both examine alterations in DNA, in some cases the Ames test was unable to detect mutagens, which later proved to be mutagenic or to interact with DNA of mammalian cells.4 Second, the concentration and exposure time were different to previous studies. Husum detected an increase in SCE formation in smoking but not in non-smoking patients exposed to anaesthetic concentrations of isoflurane and nitrous oxide.5 Unfortunately, data concerning exposure time and concentrations of anaesthetics were not available and therefore dose–responses could not be established. Although it is likely that exposure time was longer in our study, we demonstrated an increase in SCE in peripheral blood lymphocytes exposed to inhalation anaesthetics. Currently, we cannot conclude that there is any indication for the development of increased SCE rates in patients receiving clinical concentrations of isoflurane and nitrous oxide for short surgical procedures. Although the observed SCE rates were small compared with alkylating mutagens and carcinogens, it should be noted that an increase of approximately 1 SCE per cell is comparable with smoking 11–20 cigarettes a day.5 From our results, it seems unlikely that chronic exposure results in an increased SCE rate as the concentrations used in our study could not be achieved under normal operating room working conditions.6 Longterm investigations in operating room personnel exposed to different levels of waste anaesthetic gases are needed to clarify any potential genotoxicity of anaesthetic gases.
References
269
1 Reitz M, Antonini-Rumpf E, Lanz F. DNA single strand breaks in peripheral human lymphocytes after anaesthesia with isoflurane– nitrous oxide oxygen. Arzneimittelforschung 1993; 43: 1258–61 2 Boivin J. Risk of spontaneous abortion in women occupationally exposed to anaesthetic gases: a meta-analysis. Occup Environ Med 1997; 54: 541–8
Hoerauf et al.
3 Holz O, Scherer G, Brodtmeier S, et al. Determination of low level exposure to volatile aromatic hydrocarbons and genotoxic effects in workers at a styrene plant. Occup Environ Med 1995; 52: 420–8 4 Waskell L. A study of the mutagenicity of anesthetics and their metabolites. Mutat Res 1978; 57: 141–53
270
5 Husum B. Mutagenicity of inhalation anaesthetics studied by the sister chromatid exchange test in lymphocytes of patients and operating room personnel. Dan Med Bull 1987; 34: 159–70 6 Hoerauf K, Koller C, Jakob W, Taeger K, Hobbhahn J. Isoflurane waste gas exposure during general anaesthesia: the laryngeal mask compared with tracheal intubation. Br J Anaesth 1996; 77: 189–93